Calcium/sodium salt of inositol tripyrophosphate as an allosteric effector of hemoglobin

ABSTRACT

The present invention relates to mixed calcium/sodium salt of inositol tripyrophosphate, methods of preparing and methods of use. The mixed calcium/sodium salt may be a monocalcium tetrasodium salt of inositol tripyrophosphate. Methods of use include administering the above salts in an effective amount to treat diseases caused by hypoxia or other conditions associated with inadequate function of the lungs or circulatory system, such as various types of cancer and Alzheimer&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 11/497,566 filed Aug. 11, 2006, which is a continuation-in-partof U.S. application Ser. No. 11/396,338 filed Mar. 31, 2006 which is acontinuation-in-part of U.S. patent application Ser. Nos. 11/175,979filed Jul. 6, 2005, and 11/384,012 filed Mar. 17, 2006, all of which areincorporated herein by reference in their entirety. U.S. patentapplication Ser. No. 11/175,979 claims the benefit of priority to U.S.Provisional Patent Application No. 60/585,804 filed Jul. 6, 2004, whichis incorporated herein by reference in its entirety. U.S. patentapplication Ser. No. 11/384,012 claims the benefit of priority to U.S.Provisional Patent Application No. 60/663,491 filed Mar. 18, 2005, whichis incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention is directed to compositions and methods for usingthe mixed calcium/sodium salt of inositol-tripyrophosphate (ITPP-Ca/Na)to enhance oxygen delivery by red blood cells. ITPP-Ca/Na is anallosteric effector of hemoglobin which has the ability to cross theplasma membrane of red blood cells and lower the oxygen affinity of thehemoglobin of red blood cells. The present invention is further directedto the use of ITPP-Ca/Na to inhibit angiogenesis and enhance radiationsensitivity of hypoxic tumors. The present invention is further directedto the use of ITPP-Ca/Na to enhance the partial pressure of oxygen (PO₂)in hypoxic tumors.

BACKGROUND OF THE INVENTION

In the vascular system of an adult human being, blood has a volume ofabout 5 to 6 liters. Approximately one half of this volume is occupiedby cells, including red blood cells (erythrocytes), white blood cells(leukocytes), and blood platelets. Red blood cells comprise the majorityof the cellular components of blood. Plasma, the liquid portion ofblood, is approximately 90 percent water and 10 percent various solutes.These solutes include plasma proteins, organic metabolites and wasteproducts, and inorganic compounds.

The major function of red blood cells is to transport oxygen from thelungs to the tissues of the body, and transport carbon dioxide from thetissues to the lungs for removal. Very little oxygen is transported bythe blood plasma because oxygen is only sparingly soluble in aqueoussolutions. Most of the oxygen carried by the blood is transported by thehemoglobin of the erythrocytes. Erythrocytes in mammals do not containnuclei, mitochondria or any other intracellular organelles, and they donot use oxygen in their own metabolism. Red blood cells contain about 35percent by weight hemoglobin, which is responsible for binding andtransporting oxygen.

Hemoglobin is a protein having a molecular weight of approximately64,500 daltons. It contains four polypeptide chains and four hemeprosthetic groups in which iron atoms are bound in the ferrous state.Normal globin, the protein portion of the hemoglobin molecule, consistsof two alpha chains and two beta chains. Each of the four chains has acharacteristic tertiary structure in which the chain is folded. The fourpolypeptide chains fit together in an approximately tetrahedralarrangement, to constitute the characteristic quaternary structure ofhemoglobin. There is one heme group bound to each polypeptide chainwhich can reversibly bind one molecule of molecular oxygen. Whenhemoglobin combines with oxygen, oxyhemoglobin is formed. When oxygen isreleased, the oxyhemoglobin is reduced to deoxyhemoglobin.

Delivery of oxygen to tissues, including tumors, depends upon a numberof factors including, but not limited to, the volume of blood flow, thenumber of red blood cells, the concentration of hemoglobin in the redblood cells, the oxygen affinity of the hemoglobin and, in certainspecies, on the molar ratio of intraerythrocytic hemoglobins with highand low oxygen affinity. The oxygen affinity of hemoglobin depends onfour factors as well, namely: (1) the partial pressure of oxygen; (2)the pH; (3) the concentration of 2,3-diphosphoglycerate (DPG) in thehemoglobin; and (4) the concentration of carbon dioxide. In the lungs,at an oxygen partial pressure of 100 mm Hg, approximately 98% ofcirculating hemoglobin is saturated with oxygen. This represents thetotal oxygen transport capacity of the blood. When fully oxygenated, 100ml of whole mammalian blood can carry about 21 ml of gaseous oxygen.

The effect of the partial pressure of oxygen and the pH on the abilityof hemoglobin to bind oxygen is best illustrated by examination of theoxygen saturation curve of hemoglobin. An oxygen saturation curve plotsthe percentage of total oxygen-binding sites of a hemoglobin moleculethat are occupied by oxygen molecules when solutions of the hemoglobinmolecule are in equilibrium with different partial pressures of oxygenin the gas phase.

The oxygen saturation curve for hemoglobin is sigmoid. Thus, binding thefirst molecule of oxygen increases the affinity of the remaininghemoglobin for binding additional oxygen molecules. As the partialpressure of oxygen is increased, a plateau is approached at which eachof the hemoglobin molecules is saturated and contains the upper limit offour molecules of oxygen.

The reversible binding of oxygen by hemoglobin is accompanied by therelease of protons, according to the equation:

HHb⁺+O₂⇄HbO₂+H⁺

Thus, an increase in the pH will pull the equilibrium to the right andcause hemoglobin to bind more oxygen at a given partial pressure. Adecrease in the pH will decrease the amount of oxygen bound.

In the lungs, the partial pressure of oxygen in the air spaces isapproximately 90 to 100 mm Hg and the pH is also high relative to normalblood pH (up to 7.6). Therefore, hemoglobin will tend to become almostmaximally saturated with oxygen in the lungs. At that pressure and pH,hemoglobin is approximately 98 percent saturated with oxygen. On theother hand, in the capillaries in the interior of the peripheraltissues, the partial pressure of oxygen is only about 25 to 40 mm Hg andthe pH is also nearly neutral (about 7.2 to 7.3). Because muscle cellsuse oxygen at a high rate, thereby lowering the local concentration ofoxygen, the release of some of the bound oxygen to the tissue isfavored. As the blood passes through the capillaries in the muscles,oxygen will be released from the nearly saturated hemoglobin in the redblood cells into the blood plasma and then into the muscle cells.Hemoglobin will release about a fourth of its bound oxygen as it passesthrough the muscle capillaries, so that when it leaves the muscle, itwill be only about 75 percent saturated. In general, the hemoglobin inthe venous blood leaving the tissue cycles between about 65 and 97percent saturation with oxygen in its repeated circuits between thelungs and the peripheral tissues. Thus, oxygen partial pressure and pHfunction together to effect the release of oxygen by hemoglobin.

A third important factor in regulating the degree of oxygenation ofhemoglobin is the allosteric effector 2,3-diphosphoglycerate (DPG). DPGis the normal physiological effector of hemoglobin in mammalianerythrocytes. DPG regulates the oxygen-binding affinity of hemoglobin inthe red blood cells in relationship to the oxygen partial pressure inthe lungs. The higher the concentration of DPG in the cell, the lowerthe affinity of hemoglobin for oxygen.

When the delivery of oxygen to the tissues is chronically reduced, theconcentration of DPG in the erythrocytes is higher than in normalindividuals. For example, at high altitudes the partial pressure ofoxygen is significantly less. Correspondingly, the partial pressure ofoxygen in the tissues is less. Within a few hours after a normal humansubject moves to a higher altitude, the DPG level in the red blood cellsincreases, causing more DPG to be bound and the oxygen affinity of thehemoglobin to decrease. Increases in the DPG level of red cells alsooccur in patients suffering from hypoxia. This adjustment allows thehemoglobin to release its bound oxygen more readily to the tissues tocompensate for the decreased oxygenation of hemoglobin in the lungs. Thereverse change occurs when people are acclimated to high altitudes anddescend to lower altitudes.

As normally isolated from blood, hemoglobin contains a considerableamount of DPG. When hemoglobin is “stripped” of its DPG, it shows a muchhigher affinity for oxygen. When DPG is increased, the oxygen bindingaffinity of hemoglobin decreases. A physiologic allosteric effector suchas DPG is therefore essential for the normal release of oxygen fromhemoglobin in the tissues.

While DPG is the normal physiologic effector of hemoglobin in mammalianred blood cells, phosphorylated inositols are found to play the samerole in the erythrocytes of some birds and reptiles. Although inositolhexaphosphate (IHP) is unable to pass through the mammalian erythrocytemembrane, it is capable of combining with hemoglobin of mammalian redblood cells at the binding site of DPG to modify the allostericconformation of hemoglobin, the effect of which is to reduce theaffinity of hemoglobin for oxygen. For example, DPG can be replaced byIHP, which is far more potent than DPG in reducing the oxygen affinityof hemoglobin. IHP has a 1000-fold higher affinity to hemoglobin thanDPG (R. E. Benesch et al., Biochemistry, Vol. 16, pages 2594-2597(1977)) and increases the P₅₀ of hemoglobin up to values of 96.4 mm, Hgat pH 7.4, and 37 degrees C. (J. Biol. Chem., Vol. 250, pages 7093-7098(1975)).

The oxygen release capacity of mammalian red blood cells can be enhancedby introducing certain allosteric effectors of hemoglobin intoerythrocytes, thereby decreasing the affinity of hemoglobin for oxygenand improving the oxygen economy of the blood. This phenomenon suggestsvarious medical applications for treating individuals who areexperiencing lowered oxygenation of their tissues due to the inadequatefunction of their lungs or circulatory system.

Because of the potential medical benefits to be achieved from the use ofthese modified erythrocytes, various techniques have been developed inthe prior art to enable the encapsulation of allosteric effectors ofhemoglobin in erythrocytes. Accordingly, numerous devices have beendesigned to assist or simplify the encapsulation procedure. Theencapsulation methods known in the art include osmotic pulse (swelling)and reconstitution of cells, controlled lysis and resealing,incorporation of liposomes, and electroporation. Current methods ofelectroporation make the procedure commercially impractical on a scalesuitable for commercial use.

The following references describe the incorporation of polyphosphatesinto red blood cells by the interaction of liposomes loaded with IHP:Gersonde, et al., “Modification of the Oxygen Affinity of IntracellularHemoglobin by Incorporation of Polyphosphates into Intact Red BloodCells and Enhanced O₂ Release in the Capillary System”, Biblthca.Haemat., No. 46, pp. 81-92 (1980); Gersonde, et al., “Enhancement of theO₂ Release Capacity and of the Bohr-Effect of Human Red Blood Cellsafter Incorporation of Inositol Hexaphosphate by Fusion withEffector-Containing Lipid Vesicles”, Origins of Cooperative Binding ofHemoglobin (1982); and Weiner, “Right Shifting of Hb-O₂ Dissociation inViable Red Cells by Liposomal Technique,” Biology of the Cell, Vol. 47,(1983).

Additionally, U.S. Pat. Nos. 4,192,869, 4,321,259, and 4,473,563 toNicolau et al. describe a method whereby fluid-charged lipid vesiclesare fused with erythrocyte membranes, depositing their contents into thered blood cells. In this manner, it is possible to transport allostericeffectors, such as IHP into erythrocytes, where due to its much higherbinding constant IHP replaces DPG at its binding site in hemoglobin.

In accordance with the liposome technique, IHP is dissolved in aphosphate buffer until the solution is saturated and a mixture of lipidvesicles is suspended in the solution. The suspension is then subjectedto ultrasonic treatment or an injection process, and then centrifuged.The upper suspension contains small lipid vesicles containing IHP, whichare then collected. Erythrocytes are added to the collected suspensionand incubated, during which time the lipid vesicles containing IHP fusewith the cell membranes of the erythrocytes, thereby depositing theircontents into the interior of the erythrocyte. The modified erythrocytesare then washed and added to plasma to complete the product.

The drawbacks associated with the liposomal technique include poorreproducibility of the IHP concentrations incorporated in the red bloodcells and significant hemolysis of the red blood cells followingtreatment. Additionally, commercialization is not practical because theprocedure is tedious and complicated.

In an attempt to solve the drawbacks associated with the liposomaltechnique, a method of lysing and the resealing red blood cells wasdeveloped. This method is described in the following publication:Nicolau, et al., “Incorporation of Allosteric Effectors of Hemoglobin inRed Blood Cells. Physiologic Effects,” Biblthca. Haemat., No. 51, pp.92-107, (1985). Related U.S. Pat. Nos. 4,752,586 and 4,652,449 to Roparset al. also describe a procedure of encapsulating substances havingbiological activity in human or animal erythrocytes by controlled lysisand resealing of the erythrocytes, which avoids the red bloodcell-liposome interactions.

The technique is best characterized as a continuous flow dialysissystem, which functions in a manner similar to the osmotic pulsetechnique. Specifically, the primary compartment of at least onedialysis element is continuously supplied with an aqueous suspension oferythrocytes, while the secondary compartment of the dialysis elementcontains an aqueous solution which is hypotonic with respect to theerythrocyte suspension. The hypotonic solution causes the erythrocytesto lyse. The erythrocyte lysate is then contacted with the biologicallyactive substance to be incorporated into the erythrocyte. To reseal themembranes of the erythrocytes, the osmotic and/or oncotic pressure ofthe erythrocyte lysate is increased and the suspension of resealederythrocytes is recovered.

In related U.S. Pat. Nos. 4,874,690 and 5,043,261 to Goodrich et al., arelated technique involving lyophilization and reconstitution of redblood cells is disclosed. As part of the process of reconstituting thered blood cells, the addition of various polyanions, including IHP, isdescribed. Treatment of the red blood cells according to the processdisclosed results in a cell with unaffected activity. Presumably, theIHP is incorporated into the cell during the reconstitution process,thereby maintaining the activity of the hemoglobin.

In U.S. Pat. Nos. 4,478,824 and 4,931,276 to Franco et al., a secondrelated method and apparatus is described for introducing effectivelynon-ionic agents, including IHP, into mammalian red blood cells byeffectively lysing and resealing the cells. The procedure is describedas the “osmotic pulse technique.” In practicing the osmotic pulsetechnique, a supply of packed red blood cells is suspended and incubatedin a solution containing a compound which readily diffuses into and outof the cells, the concentration of the compound being sufficient tocause diffusion thereof into the cells so that the contents of the cellsbecome hypertonic. Next, a trans-membrane ionic gradient is created bydiluting the solution containing the hypertonic cells with anessentially isotonic aqueous medium in the presence of at least onedesired agent to be introduced, thereby causing diffusion of water intothe cells with a consequent swelling and an increase in permeability ofthe outer membranes of the cells. This “osmotic pulse” causes thediffusion of water into the cells and a resultant swelling of the cellswhich increase the permeability of the outer cell membrane to thedesired agent. The increase in permeability of the membrane ismaintained for a period of time sufficient only to permit transport ofat least one agent into the cells and diffusion of the compound out ofthe cells.

Polyanions which may be used in practicing the osmotic pulse techniqueinclude pyrophosphate, tripolyphosphate, phosphorylated inositols,2,3-diphosphoglycerate (DPG), adenosine triphosphate, heparin, andpolycarboxylic acids which are water-soluble, and non-disruptive to thelipid outer bilayer membranes of red blood cells.

The osmotic pulse technique has several shortcomings including low yieldof encapsulation, incomplete resealing, loss of cell content and acorresponding decrease in the life span of the cells. The technique istedious, complicated and unsuited to automation. For these reasons, theosmotic pulse technique has had little commercial success.

Another method for encapsulating various biologically-active substancesin erythrocytes is electroporation. Electroporation has been used forencapsulation of foreign molecules in different cell types, includingIHP in red blood cells, as described in Mouneimne, et al., “Stablerightward shifts of the oxyhemoglobin dissociation curve induced byencapsulation of inositol hexaphosphate in red blood cells usingelectroporation,” FEBS, Vol. 275, No. 1, 2, pp. 117-120 (1990). Also,see U.S. Pat. No. 5,612,207.

Angiogenesis is the generation of new blood vessels into a tissue ororgan and is related to oxygen tension in the tissues. Under normalphysiological conditions, humans and animals undergo angiogenesis onlyin very specific, restricted situations. For example, angiogenesis isnormally observed in wound healing, fetal and embryonal development, andformation of the corpus luteum, endometrium and placenta.

Angiogenesis is controlled through a highly regulated system ofangiogenic stimulators and inhibitors. The control of angiogenesis isaltered in certain disease states and, in many cases, pathologicaldamage associated with the diseases is related to uncontrolledangiogenesis. Both controlled and uncontrolled angiogenesis are thoughtto proceed in a similar manner. Endothelial cells and pericytes,surrounded by a basement membrane, form capillary blood vessels.Angiogenesis begins with the erosion of the basement membrane by enzymesreleased by endothelial cells and leukocytes. Endothelial cells, liningthe lumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating a new blood vessel.

Persistent, unregulated angiogenesis occurs in many disease states,tumor metastases, and abnormal growth by endothelial cells. The diversepathological disease states in which unregulated angiogenesis is presenthave been grouped together as angiogenic-dependent orangiogenic-associated diseases.

The hypothesis that tumor growth is angiogenesis-dependent was firstproposed in 1971. (Folkman, New Eng. J. Med., 285:1182-86 (1971)). Inits simplest terms, this hypothesis states: “Once tumor ‘take’ hasoccurred, every increase in tumor cell population must be preceded by anincrease in new capillaries converging on the tumor.” Tumor ‘take’ iscurrently understood to indicate a prevascular phase of tumor growth inwhich a population of tumor cells occupying a few cubic millimetersvolume, and not exceeding a few million cells, can survive on existinghost microvessels. Expansion of tumor volume beyond this phase requiresthe induction of new capillary blood vessels. For example, pulmonarymicrometastases in the early prevascular phase in mice would beundetectable except by high power microscopy on histological sections.

Angiogenesis has been associated with a number of different types ofcancer, including solid tumors and blood-borne tumors. Solid tumors withwhich angiogenesis has been associated include, but are not limited to,rhabdomyosarcomas, retinoblastoma, Ewing's sarcoma, neuroblastoma, andosteosarcoma. Angiogenesis is also associated with blood-borne tumors,such as leukemias, any of various acute or chronic neoplastic diseasesof the bone marrow in which unrestrained proliferation of white bloodcells occurs, usually accompanied by anemia, impaired blood clotting,and enlargement of the lymph nodes, liver and spleen. It is believedthat angiogenesis plays a role in the abnormalities in the bone marrowthat give rise to leukemia tumors and multiple myeloma diseases.

One of the most frequent angiogenic diseases of childhood is thehemangioma. A hemangioma is a tumor composed of newly formed bloodvessels. In most cases, the tumors are benign and regress withoutintervention. In more severe cases, the tumors progress to largecavernous and infiltrative forms and create clinical complications.Systemic forms of hemangiomas, hemangiomatoses, have a high mortalityrate. Therapy-resistant hemangiomas exist that cannot be treated withtherapeutics currently in use.

Another angiogenesis associated disease is rheumatoid arthritis. Theblood vessels in the synovial lining of the joints undergo angiogenesis.In addition to forming new vascular networks, the endothelial cellsrelease factors and reactive oxygen species that lead to pannus growthand cartilage destruction. Angiogenesis may also play a role inosteoarthritis. The activation of the chondrocytes by angiogenic-relatedfactors contributes to the destruction of the joint. At a later stage,the angiogenic factors promote new bone growth. Therapeutic interventionthat prevents the cartilage destruction could halt the progress of thedisease and provide relief for persons suffering with arthritis.

Chronic inflammation may also involve pathological angiogenesis. Suchdiseases as ulcerative colitis and Crohn's disease show histologicalchanges with the ingrowth of new blood vessels into inflamed tissues.Bartonelosis, a bacterial infection found in South America, can resultin a chronic stage that is characterized by proliferation of vascularendothelial cells. Another pathological role associated withangiogenesis is found in atherosclerosis. The plaques formed within thelumen of blood vessels have been shown to have angiogenic stimulatoryactivity.

As mentioned above, several lines of evidence indicate that angiogenesisis essential for the growth and persistence of solid tumors and theirmetastases. Once angiogenesis is stimulated, tumors upregulate theproduction of a variety of angiogenic factors, including fibroblastgrowth factors (aFGF and bFGF) and vascular endothelial growthfactor/vascular permeability factor (VEGF/VPF) [2,3].

The role of VEGF in the regulation of angiogenesis has been the objectof intense investigation [5-10]. Whereas VEGF represents a critical,rate-limiting step in physiological angiogenesis, it appears to be alsoimportant in pathological angiogenesis, such as that associated withtumor growth [11]. VEGF is also known as vascular permeability factor,based on its ability to induce vascular leakage [13]. Several solidtumors produce ample amounts of VEGF, which stimulates proliferation andmigration of endothelial cells, thereby inducing neovascularization[12,13]. VEGF expression has been shown to significantly affect theprognosis of different kinds of human cancer. Oxygen tension in thetumor has a key role in regulating the expression of VEGF gene. VEGFmRNA expression is induced by exposure to low oxygen tension under avariety of pathophysiological circumstances [13]. Growing tumors arecharacterized by hypoxia, which induces expression of VEGF and may alsobe a predictive factor for the occurrence of metastatic disease.

What is needed, therefore, is a substantially non-toxic composition andmethod that can regulate oxygen tension in the tissue, especially atumor. In addition, what is needed is a simple and easily administered,preferably orally, composition that is capable of causing significantright shifts of the P₅₀ value for red blood cells.

SUMMARY OF THE INVENTION

The present invention provides a composition comprising the mixedcalcium/sodium salt of inositol-tripyrophosphate (ITPP-Ca/Na) that iseffective in treating diseases characterized by abnormal angiogenesis.The compositions and methods of the present invention have a distinctadvantage over the prior art in that the compositions and methods of thepresent invention are substantially non-toxic and demonstrate improvedsolubility when compared to compositions in the prior art.

The present invention also comprises a pharmaceutical compositioncomprising the calcium/sodium salt of ITPP and a pharmaceuticallyacceptable adjuvant, diluent, carrier, or excipient thereof. In thispharmaceutical composition, the inositol tripyrophosphate is optimallymyo-inositol 1,6:2,3:4,5 tripyrophosphate. In an alternate embodimentthe composition may comprise the monocalcium tetrasodium salt ofmyo-inositol 1,6:2,3:4,5 tripyrophosphate.

The present invention also provides for substantially non-toxic methodsof using the above compositions of ITPP-Ca/Na for increasing theregulated delivery of oxygen to tissues including tumors. For example,the regulation of vascular endothelial growth factor (VEGF) in a humanor animal can be effected using ITPP-Ca/Na which has entered the redblood cell, thus lowering the affinity for oxygen of circulatingerythrocytes. In an embodiment of the present invention, ITPP-Ca/Na canaffect VEGF mRNA expression, protein concentration, and tumor cellproliferation. Also, a method of regulating VEGF expression, both invitro and in vivo, using ITPP-Ca/Na is contemplated and therefore withinthe scope of the present invention.

The present invention further comprises methods for using the abovecompositions of ITPP-Ca/Na in pure hemoglobin and in red blood cells todeliver oxygen to solid tumors, to inhibit angiogenesis and to enhanceradiation sensitivity of hypoxic tumors. The present invention isfurther directed to the use of ITPP-Ca/Na to enhance PO₂ in hypoxictumors. ITPP-Ca/Na is an allosteric effector of hemoglobin and iscapable of reducing hemoglobin's affinity for oxygen, which enhances therelease of oxygen by hemoglobin. Upon cellular demand, ITPP-Ca/Na caninhibit VEGF expression in tumor cells and, thus, angiogenesis.

A disease characterized by undesirable angiogenesis or undesirableangiogenesis, as defined herein includes, but is not limited to,excessive or abnormal stimulation of endothelial cells (e.g.atherosclerosis), blood borne tumors, solid tumors and tumor metastasis,benign tumors, for example, hemangiomas, acoustic neuromas,neurofibromas, trachomas, and pyogenic granulomas, vascularmalfunctions, abnormal wound healing, inflammatory and immune disorders,Bechet's disease, gout, or gouty arthritis, diabetic retinopathy andother ocular angiogenic diseases such as retinopathy of prematurity(retrolental fibroplasic), macular degeneration, corneal graftrejection, neovascular glaucoma and Osler Weber syndrome(Osler-Weber-Rendu disease). Cancers that can be treated by the presentinvention include, but is not limited to, breast cancer, prostratecancer, renal cell cancer, brain cancer, ovarian cancer, colon cancer,bladder cancer, pancreatic cancer, stomach cancer, esophageal cancer,cutaneous melanoma, liver cancer, lung cancer, testicular cancer, kidneycancer, bladder cancer, cervical cancer, lymphoma, parathyroid cancer,penile cancer, rectal cancer, small intestine cancer, thyroid cancer,uterine cancer, Hodgkin's lymphoma, lip and oral cancer, skin cancer,leukemia or multiple myeloma.

An object of the invention is to provide a substantially non-toxiccomposition and method for treating cancer and other angiogenic diseasestates and conditions using ITPP-Ca/Na in an effective dose.

Another object of the invention is to provide a composition and methodfor enhancing oxygen delivery to hypoxic tumors using ITPP-Ca/Na in aneffective dose.

Yet another object of the invention is to provide a composition andmethod for inhibiting angiogenesis using ITPP-Ca/Na in an effectivedose.

A further object of the invention is to provide a composition and methodfor enhancing radiation sensitivity of hypoxic tumors using ITPP-Ca/Nain an effective dose.

It is yet another object of the invention to provide a composition andmethod of treating hypoxic tumors and diseases using ITPP-Ca/Na in aneffective dose.

Another object of the invention is to provide a composition and methodusing ITPP-Ca/Na in an effective dose that can regulate oxygen tensionin the tissue, especially a tumor.

A further object of the invention is to provide a simple and easilyadministered, preferably oral, composition that is capable of causingsignificant right shifts of the P₅₀ value for red blood cells usingITPP-Ca/Na in an effective dose.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows ITPP-Ca/Na loaded red blood cell suppression of HIF-1induction, VEGF and angiogenesis of hypoxic endothelial cells in vitro.

FIG. 2 shows the potential of ITPP-Ca/Na as a dual action radiationsensitizer and angiogenesis inhibitor in pancreatic and rectal cancers.

FIG. 3 shows an agarose gel indicating the VEGF mRNA concentrations intumors from control and ITPP drinking animals.

FIG. 4 show a Western blot assay of expressed VEGF in tumors of controland ITPP-treated Lewis Lung carcinoma (LLC) tumor-bearing animals.

FIG. 5 shows a synthesis scheme for synthesizing the monocalciumtetrasodium salt of myo-inositol 1,6:2,3:4,5 tripyrophosphate.

FIG. 6 shows an oxygen fixation curve for hemoglobin after incubationwith ITPP-Ca4Na.

FIG. 7 shows the percent shift of the p50 of whole human blood afterincubation with ITPP-Ca4Na.

DETAILED DESCRIPTION OF THE INVENTION

Compositions that are useful in accordance with the present inventioninclude the mixed calcium/sodium salt of inositol-tripyrophosphate(ITPP-Ca/Na). ITPP exhibits anti-angiogenic and anti-tumor properties,and is useful in controlling angiogenesis-, or proliferation-relatedevents, conditions or substances. As used herein, the control of anangiogenic-, or proliferation-related event, condition, or substancerefers to any qualitative or quantitative change in any type of factor,condition, activity, indicator, chemical or combination of chemicals,mRNA, receptor, marker, mediator, protein, transcriptional activity orthe like, that may be or is believed to be related to angiogenesis orproliferation, and that results from administering the composition ofthe present invention. Those skilled in the art will appreciate that theinvention extends to other compositions or compounds in the claimsbelow, having the described characteristics. These characteristics canbe determined for each test compound using the assays detailed below andelsewhere in the literature.

Other such assays include counting of cells in tissue culture plates orassessment of cell number through metabolic assays or incorporation intoDNA of labeled (radiochemically, for example ³H-thymidine, orfluorescently labeled) or immuno-reactive (BrdU) nucleotides. Inaddition, antiangiogenic activity may be evaluated through endothelialcell migration, endothelial cell tubule formation, or vessel outgrowthin ex-vivo models, such as rat aortic rings.

When administered orally, ITPP exhibits anti-tumor andanti-proliferative activity with little or no toxicity. ITPP was testedfor its ability to induce a decrease of the O₂-affinity of hemoglobinmeasured as a shift of the P₅₀ value (P₅₀ at 50% saturation ofhemoglobin). With murine hemoglobin and whole blood, P₅₀ shifts tohigher PO₂ of up to 250% with hemoglobin and up to 40% with whole bloodwere observed.

The results obtained with ITPP in mice and pigs strongly suggest thepossibility of its development as a therapeutic, due to its ability toenhance, in a regulated manner, oxygen delivery by red blood cells inthe cases of blood flow impairment.

It has been found that pigs injected intravenously with ITPP-Na at arate of 1 g/kg weight had beneficial properties associated with theintroduction of ITPP-Na into their systems (as described in U.S.Provisional Patent Application 60/585,804, which is herein incorporatedby reference in its entirety); however, the introduction of ITPP-Na alsoresulted in a number of adverse side effects. These side effectsincluded flushing, an increase in the heart rate, and a decrease in theCa²⁺ plasma concentration. Therefore a less toxic form of ITPP thatmaintains a good solubility profile is needed.

ITPP, when administered orally, intravenously, or intraperitoneally,inhibits angiogenesis in growing tumors by enhancing PO₂ in the formingtumors. This invention further provides for methods of regulation ofvascular endothelial growth factor (VEGF) in a human or animal, byadministering to the human or animal an effective amount of ITPP. Moreparticularly, this invention provides for dose-dependent effects of ITPPon VEGF mRNA and protein expressions in the LLC cell line. VEGF geneexpression in tumor bearing C57BL/6 mice was assayed and the effects ofITPP-induced down regulation of VEGF have been determined and correlatedwith modulation of cell proliferation. This invention resulted in thedevelopment of methods to control VEGF mRNA expression, proteinconcentration, and tumor cell proliferation. The results of thesestudies indicate a strong correlation between dose-dependentITPP-induced down regulation of VEGF and cellular proliferation andsuggests that ITPP can reduce VEGF mediated tumor angiogenesis, as wellas the rate of tumor cell proliferation. Thus, down-regulation of VEGFby ITPP decreases tumor cell proliferation.

The shifting of the P₅₀ value to higher O₂-partial pressures inhibitsthe expression of the hypoxia gene encoding VEGF in the tumors.Expression of the hypoxia gene encoding VEGF is necessary forangiogenesis to be stimulated in tumors. If this does not occur,angiogenesis is seriously inhibited and new vessels are not formed intumors.

The results obtained concerning VEGF expression suggests that oxygenpartial pressure in tumors is elevated upon administration of ITPP, asthis elevation is the cause of inhibition of expression of this hypoxiagene. This observation raises a very important question, namely, whetherthis enhancement of PO₂ may act as a powerful radiosensitizer of cancercells. Oxygen is a very potent radiosensitizer and, if indeed PO₂ in thetumors is enhanced by ITPP, this may have major consequences inenhancing the efficacy of radiation therapy of cancer.

ITPP is a potential significant adjuvant in the therapy of solid tumorsas inhibitor of angiogenesis on one hand, and as a radiosensitizer onthe other.

It is known that medial temporal oxygen metabolism is markedly affectedin patients with mild-to-moderate Alzheimer's disease. This measuresubstantiated the functional impairment of the medial temporal region inAlzheimer's disease. It also known that mean oxygen metabolism in themedial temporal, as well as in the parietal and lateral temporalcortices is significantly lower in the patients that are shown to haveAlzheimer's disease than in control groups without Alzheimer's disease(see Ishii et al., J. Nucl Med. 37(7):1159-65, July 1996, which isherein incorporated by reference in its entirety). Thus, one potentialmeans of treating patients shown to have Alzheimer's disease is toincrease oxygen across the blood brain barrier. One method of doing sowould be to use an allosteric effector of hemoglobin such as treatmentwith ITPP, such as with the calcium/sodium salt of ITPP.

The use of ITPP, such as with the calcium/sodium salt of ITPP, may alsohelp in the treatment of a variety of vascular diseases associated withvarious forms of dementia. Because the brain relies on a network ofvessels to bring it oxygen-bearing blood, if the oxygen supply to thebrain fails, brain cells are likely to die and this can cause symptomsof vascular dementia. These symptoms can occur either suddenly,following a stroke, or over time through a series of small strokes.Thus, one potential means of treating patients with vascular diseasesassociated with various forms of dementia is to increase the oxygenavailable to affected areas such as across the blood brain barrier. Onemethod of doing so would be to use an allosteric effector of hemoglobinsuch as treatment with ITPP, such as with the calcium/sodium salt ofITPP.

Moreover, treatment of an individual with an allosteric effector ofhemoglobin such as the calcium/sodium salt of ITPP may have beneficialeffects for both stroke victims and osteoporosis. Although stroke andthe bone-thinning disease osteoporosis are usually thought of as twodistinct health problems, it has been found that there may be aconnection between them. Patients who survive strokes are significantlymore likely to suffer from osteoporosis, a disease that puts them athigh risk for bone fractures. Often, the fractures in stroke patientsoccur on the side of the body that has been paralyzed from the stroke.

It is known that a stroke occurs when the supply of blood and oxygen tothe brain ceases or is greatly reduced. If a portion of the brain losesits supply of nutrient-rich blood and oxygen, the bodily functionscontrolled by that part of the brain (vision, speech, walking, etc.) areimpaired. Annually, more than 500,000 people in the United States sufferstrokes and 150,000 of those people die as a result thereof. One meansof increasing oxygen flow to the brain is by use of an allostericeffector of hemoglobin such as treatment with the calcium/sodium salt ofITPP. Accordingly, a potential method of treating individuals who mightpotentially suffer stroke or osteoporosis is by treatment of anindividual with, for example, the calcium/sodium salt of ITPP.

Also contemplated by the present invention are implants or other devicescomprised of the compounds or drugs of ITPP, or prodrugs thereof, wherethe drug or prodrug is formulated in a biodegradable ornon-biodegradable polymer for sustained release. Non-biodegradablepolymers release the drug in a controlled fashion through physical ormechanical processes without the polymer itself being degraded.

Biodegradable polymers are designed to gradually be hydrolyzed orsolubilized by natural processes in the body, allowing gradual releaseof the admixed drug or prodrug. The drug or prodrug can be chemicallylinked to the polymer or can be incorporated into the polymer byadmixture. Both biodegradable and non-biodegradable polymers and theprocess by which drugs are incorporated into the polymers for controlledrelease are well known to those skilled in the art. Examples of suchpolymers can be found in many references, such as Brem et al., J.Neurosurg 74: pp. 441-446 (1991), which is herein incorporated byreference in its entirety. These implants or devices can be implanted inthe vicinity where delivery is desired, for example, at the site of atumor.

In addition to the compounds of the present invention, thepharmaceutical composition of this invention may also contain, or beco-administered (simultaneously or sequentially) with, one or morepharmacological agents of value in treating one or more diseaseconditions referred to hereinabove.

A person skilled in the art will be able by reference to standard texts,such as Remington's Pharmaceutical Sciences 17^(th) edition, todetermine how the formulations are to be made and how these may beadministered.

In a further aspect of the present invention there is provided use ofcompounds of ITPP, such as ITPP-Ca/Na or prodrugs thereof, according tothe present invention for the preparation of a medicament for theprophylaxis or treatment of conditions associated with angiogenesis oraccelerated cell division or inflammation.

In a further aspect of the present invention there is provided apharmaceutical composition comprising compounds of ITPP, such asITPP-Ca/Na or prodrugs thereof, according to the present invention,together with a pharmaceutically acceptable carrier, diluent, adjuvantor excipient.

The pharmaceutical composition may be used for the prophylaxis ortreatment of conditions associated with angiogenesis or accelerated celldivision or inflammation, for treatment of Alzheimer's disease,treatment of stroke and/or osteoporosis.

In a still further aspect of the present invention there is provided amethod of prophylaxis or treatment of a condition associated withangiogenesis or accelerated or increased amounts of cell division,hypertrophic growth, or inflammation, said method includingadministering to a patient in need of such prophylaxis or treatment aneffective amount of compounds of ITPP, such as ITPP-Ca/Na or prodrugsthereof, according to the present invention, as described herein. Itshould be understood that prophylaxis or treatment of said conditionincludes amelioration of said condition.

By “an effective amount” as referred to in this specification, it ismeant a therapeutically or prophylactically effective amount. Suchamounts can be readily determined by an appropriately skilled person,taking into account the condition to be treated, the route ofadministration and other relevant factors. Such a person will readily beable to determine a suitable dose, mode and frequency of administration.“Individual” as referred to in this application refers to any animalthat may be in need of treatment for a given condition. “Individual”includes humans, other primates, household pets, livestock, rodents,other mammals, and any other animal(s) that may typically be treated bya veterinarian.

The compositions described above can be provided as physiologicallyacceptable formulations using known techniques, and these formulationscan be administered by standard routes. In general, the combinations maybe administered by the topical, oral, rectal, intraperitoneal orparenteral (e.g., intravenous, subcutaneous or intramuscular) route. Inaddition, the combinations may be incorporated into polymers allowingfor sustained release, the polymers being implanted in the vicinity ofwhere delivery is desired, for example, at the site of a tumor, or intoan a cavity or blood vessel that will lead to easy delivery to the placeto be treated. The dosage of the composition will depend on thecondition being treated, the particular derivative used, and otherclinical factors such as weight and condition of the patient and theroute of administration of the compound. However, for oraladministration, a recommended dosage is in the range of 0.1 to 5.0g/kg/day. A dosage for oral administration is in the range of 0.5 to 2.0g/kg/day or alternatively, about 0.5 to about 1.5 g/kg/day. In analternate embodiment, a dosage for oral administration is in the rangeof about 0.80 to 1.0 g/kg/day or alternatively, about between 0.9 to 1.1g/kg/day.

The formulations in accordance with the present invention can beadministered in the form of tablet, a capsule, a lozenge, a cachet, asolution, a suspension, an emulsion, a powder, an aerosol, asuppository, a spray, a pastille, an ointment, a cream, a paste, a foam,a gel, a tampon, a pessary, a granule, a bolus, a mouthwash, or atransdermal patch.

The formulations include those suitable for oral, rectal, nasal,inhalation, topical (including dermal, transdermal, buccal andsublingual), vaginal, parenteral (including subcutaneous, intramuscular,intravenous, intraperitoneal, intradermal, intraocular, intratracheal,and epidural) or inhalation administration. The formulations mayconveniently be presented in unit dosage form and may be prepared byconventional pharmaceutical techniques. Such techniques include the stepof bringing into association the active ingredient and a pharmaceuticalcarrier(s) or excipient(s). In general, the formulations are prepared byuniformly and intimately bringing into association the active ingredientwith liquid carriers or finely divided solid carriers or both, and then,if necessary, shaping the product.

Formulations contemplated as part of the present invention includenanoparticles formulations made by methods disclosed in U.S. patentapplication Ser. No. 10/392,403 (Publication No. 2004/0033267) which ishereby incorporated by reference in its entirety. By formingnanoparticles, the compositions disclosed herein are shown to haveincreased bioavailability. Preferably, the particles of the compounds ofthe present invention have an effective average particle size of lessthan about 2 microns, less than about 1900 nm, less than about 1800 nm,less than about 1700 nm, less than about 1600 nm, less than about 1500nm, less than about 1400 nm, less than about 1300 nm, less than about1200 nm, less than about 1100 nm, less than about 1000 nm, less thanabout 900 nm, less than about 800 run, less than about 700 nm, less thanabout 600 nm, less than about 500 nm, less than about 400 nm, less thanabout 300 nm, less than about 250 nm, less than about 200 nm, less thanabout 150 nm, less than about 100 nm, less than about 75 nm, or lessthan about 50 nm, as measured by light-scattering methods, microscopy,or other appropriate methods well known to those of ordinary skill inthe art.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil emulsion, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide a slow or controlled release of theactive ingredient therein.

Formulations suitable for topical administration in the mouth includelozenges comprising the ingredients in a flavored basis, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the ingredient to be administered in a suitableliquid carrier.

Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels and pastes comprising theingredient to be administered in a pharmaceutically acceptable carrier.A preferred topical delivery system is a transdermal patch containingthe ingredient to be administered.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter and/or asalicylate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is taken; i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose.Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining, in addition to the active ingredient, ingredients such ascarriers as are known in the art to be appropriate.

Formulation suitable for inhalation may be presented as mists, dusts,powders or spray formulations containing, in addition to the activeingredient, ingredients such as carriers as are known in the art to beappropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example, sealed ampules and vials, and may be stored infreeze-dried (lyophilized) conditions requiring only the addition of asterile liquid carrier, for example, water for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions may beprepared from sterile powders, granules and tablets of the kindspreviously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient.

It should be understood that in addition to the ingredients,particularly those mentioned above, the formulations of the presentinvention may include other agents conventional in the art having regardto the type of formulation in question, for example, those suitable fororal administration may include flavoring agents or other agents to makethe formulation more palatable and more easily swallowed.

EXPERIMENTAL

For the in vitro experiments, ITPP was dissolved in deionized water, pHwas adjusted at pH 7 and, for incubation with whole blood, theosmolarity of the ITPP solutions was adjusted with glucose to 270-297mOsM. Mixtures of hemoglobin and ITPP were measured with a HEMOXanalyzer (PD Marketing, London) immediately after mixing. Red bloodcells were incubated with ITPP for 1 hour at 37° C. Followingincubation, the cells were washed 3 times with Bis-Tris-buffer (pH=7.0)and then used for P₅₀ measurement.

In experiments conducted in vivo in which ITPP was administered orally,a significant shift of the P₅₀ value of circulating RBCs was observed.ITPP was dissolved in drinking water at a 20 g/L-concentration (0.27 mM,pH ˜7.0.) and offered for drinking ad libitum.

The following examples illustrate but do not limit the invention. Thus,the examples are presented with the understanding that modifications maybe made and still be within the spirit and scope of the invention.

Example 1 Effectiveness of the Mixed Calcium Sodium Salt of myo-InositolTripyrophosphate

When myo-inositol tripyrophosphate-sodium salt (ITPP-Na) is mixed withCaCl₂, a mixture of ITPP-Na (myo-inositol tripyrophosphate-sodium salt)and ITPP-Ca (myo-inositol tripyrophosphate-calcium salt) is obtained.This mixture, when added to free hemoglobin or to whole blood induces aP₅₀ shift of 170% and 25%, respectively as shown in Tables 2 and 3below. Please see the results in Tables 2 and 3 for compound 15. Thecompounds in Tables 2 and 3 are as follows: 4 is the pyridinium salt ofITPP, 5 is the sodium salt of ITPP (i.e., ITPP-Na), 7 is theN,N-dimethylcyclohexyl ammonium salt of ITPP, 11 is the cycloheptylammonium salt of ITPP, 12 is the cyclooctyl ammonium salt of ITPP, 13 isthe piperazinium salt of ITPP, 14 is the tripiperazinium salt of ITPP,and 15 is the calcium salt of ITPP (i.e., ITPP-Ca).

In Tables 2 and 3, the effectiveness of all of the salts of ITPPregarding their ability to act as allosteric effectors of hemoglobin canbe seen. The sodium salt and the calcium salt of ITPP appear to be thebest allosteric effectors for both free hemoglobin (Table 2) and inwhole blood (Table 3). However, pigs injected intravenously with ITPP-Naat a rate of 1 g/kg weight resulted in a number of adverse side effects.The intravenous injection of pigs with ITPP-Na resulted in flushing, anincrease in the heart rate, and a decrease in the Ca²⁺ plasmaconcentration from 2.38 mmol/L to 1.76 mmol/L.

Administration of the mixture of the sodium and calcium salt of ITPP, atthe same dosage did not induce any of the cited effects and the Ca²⁺plasma concentration stayed unchanged at 2.38 mmol/L.

FIG. 1 shows the ability of ITPP-Ca/Na loaded red blood cellssuppression of HIF-1 induction, VEGF and angiogenesis of hypoxicendothelial cells in vitro. FIG. 2 demonstrates the potential ofITPP-Ca/Na as a radiation sensitizer and as an angiogenesis inhibitor inpancreatic and rectal cancers.

TABLE 2 P₅₀ values of free Hb after incubation with compounds 4, 5, 7,11-14 and 15, in vitro P₅₀ (Torr) P₅₀ (Torr) P₅₀ increase Compound FreeHb Hb + compound (%) + SD 4 (H) 15.3 31.6 107 ± 22 (M) 25.0 50.0 100 ±18 5 (H) 15.3 49.8 225 ± 19 (M) 24.9 69.7 180 ± 25 (P) 22.0 68.1 209 ±39 7 (M) 24.9 45.1  81 ± 15 11 (M) 24.9 43.8 71 ± 3 12 (M) 24.9 30.6 23± 5 13 (M) 23.4 67.7 189 ± 43 14 (M) 23.4 82.9 254 ± 49 15 (H) 123 33.1170 ± 32 (M) 26.9 61.9 130 ± 30 H = human; M = murine; P = porcine freeHb. Concentration of the compound solution was 60 mM; means of P₅₀shifts in % are shown. SD = standard deviation. Compounds 4, 7, 11, 12,14 and 15: three P₅₀ values each were used for the calculation of means;compound 5: with human blood: five values, murine blood: ten values andporcine blood; three values were used for the calculation of the meansof P₅₀ shifts in %.

TABLE 3 P₅₀ values of whole blood after incubation with compounds 4, 5,7, 11-14 and 15, in vitro P₅₀ (Torr) P₅₀ (Torr) compound + P₅₀ increaseCompound whole blood whole blood (%) + SD 4 (H) 22.1 24.3 10 ± 4 (M)37.9 42.7 13 ± 2 5 (H) 22.1 30.8 39^(a) ± 5   (P) 31.6 44.2 40^(a) ± 3  (M) 36.7 47.4 29^(b) ± 3  7 (M) 40.1 52.0 30 ± 3 11 (M) 37.9 45.5 20 ± 212 (M) 37.9 41.3  9 ± 1 13 (M) 37.9 41.7 10 ± 2 14 (M) 39.2 41.9  7 ± 115 (M) 39.2 42.3  8 ± 2 (H) 24.8 31.0 25 ± 3 (M) 40.1 55.3 38^(a) ± 4  H = human; M = murine; P = porcine whole blood. Compound concentrations:30 mM; means of (four single values) P₅₀ shifts □ SD are shown.^(a)Compound concentration: 60 mM. ^(b)Compound concentration: 4 mM.

Example 2 Effect of In Vivo Lowering of Hemoglobin's Affinity for O₂ byITPP on Intratumoral PO₂ Angiogenesis and Expression of VEGF mRNA

ITPP, when administered orally, intravenously, or intraperitoneally,inhibits angiogenesis in growing tumors by enhancing the PO₂ in theforming tumors. Thirty (30) C57BL/6 mice received 20 g/L of ITPP orallyuntil the P₅₀ value showed a shift of at least 20% above the controlvalue. Thereafter, all animals received 1×10⁶ Lewis Lung carcinoma (LLC)cells, injected in the dorsal cavity. At different time points, the VEGFmRNA were assayed by RT-PCR in the tumors growing in both groups ofmice.

Tumor tissue samples were ground in a RIPA lysis buffer (1% Nonidet p-40detergent, 50 mM Tris pH 8.0, 137 mM NaCl, 10% glycerol) supplementedwith protease inhibitor cocktail (Roche, Reinach, Switzerland). Aftercentrifugation for 10 minutes at 4° C. and 12,000 g, proteinconcentrations of tissue extracts were determined according to theBradford method. Detergent soluble protein samples (10 mg) wereseparated by size on a SDS-PAGE in 10% acrylamide gels and transferredto nitrocellulose membrane (Protran BA 85, Schleicher and Schuell,Dassel, Germany). Membranes were blocked for 3 hours at room temperaturein 10% skim milk in Tris buffer saline containing 0.1% Tween, before anovernight incubation at 4° C. with rabbit polyclonal antibodiesrecognizing human, mouse and rat vascular endothelial growth factor(VEGF A-20, sc-152, Santa Cruz Biotechnology, Santa Cruz, Calif.) at adilution of 1:200. Membranes were then probed for primary antibody withanti-rabbit (1:16,000) peroxidase conjugates (Sigma-Aldrich, L′Isled′Abeau Chesnes, France) for 60 minutes at room temperature. Theresulting complexes were visualized by enhanced chemiluminescenceautoradiography (Amersham Pharma Biotech, Orsay, France).

There was a difference in the level of mRNA of the VEGF gene in bothgroups. FIG. 3 shows an agarose gel indicating the VEGF mRNAconcentrations in tumors from control and ITPP drinking animals. TheRT-PCR agarose gel assay of VEGF mRNAs from tumor tissue taken from 2mice each on day 15 after inoculation of LLC cells (track 1: controls,track 2: ITPP treated animals) and day 30 after inoculation (track 3:control animals, track 4: ITPP treated animals). FIG. 4 shows theWestern blot assay of the expressed VEGF in tumors of control andITPP-treated LLC tumor-bearing animals.

Quantification of the gel assays indicated a reduction by a factor of10,000 of the amount of VEGF mRNAs detected in the tumors of animalshaving received ITPP, at day 9 and then, while differences remainbetween treated and untreated animals, they tend to decrease. Thisindicates that ITPP taken up by circulating red blood cellssignificantly increases tumor PO₂.

Example 3 Method of Synthesizing Monocalcium Tetrasodium myo-InositolTripyrophosphate Materials:

1. myo-Inositol hexakisphosphate dodecasodium salt (Product Number:P0109, Sigma).

2. Dicyclohexylcarbodiimide (Product Number: D80002, Aldrich). 3.Triethylamine (Product Number: 15791, Acros)

4. Dowex 50Wx8 hydrogen form (Product Number: 217506, Aldrich).

5. Ca(OH)₂ (Product Number: 239232, Aldrich). 6. NaOH (Product Number:1040017, Sds). 7. Acetonitrile (Product Number: 34851, Aldrich). 8.Deionized Water. Procedure:

The following synthesis scheme is shown in FIG. 5. Dowex 50WX8-200 ionexchange resin (800 g) was washed with water until the elute wascolorless. myo-Inositol hexakisphosphate dodecasodium salt (note-1) (100g, 0.108 mol, 1.0 eq) was added portionwise (10 g/portion in about 45minutes) to 500 mL of water. Each portion was dissolved with stirring atroom temperature (23° C.) before the next portion was added. Thissolution was then passed through the column containing the above washedDowex 50WX8-200 ion exchange resin and eluted with water (4×200 mL) toobtain the free phytic acid (note-2). To the combined acidic fractions,triethylamine (400 mL, 2.87 mol, 26.5 eq, about twice the theoreticalquantity) was added over 1 to 2 minutes at room temperature (23° C.) andthe mixture was stirred vigorously for 15 minutes (note-3). Then thesolvent was evaporated on a rotary evaporator (60° C., 68-22 mbar)(note-4) and the residue was dried under high vacuum for 1 hr at roomtemperature (23° C.) to give the hexatriethylammonium myo-inositolhexakisphosphate (note-5).

To this hexatriethylammonium myo-inositol hexakisphosphate dissolved inwater (800 mL), dicyclohexylcarbodiimide (142 g, 0.68 mol, 6.3 eq)dissolved in acetonitrile (1600 mL) was added at once and the mixturewas refluxed for 12 h (note-6). One more equivalent ofdicyclohexylcarbodiimide (22 g, 0.108 mol, 1.0 eq) dissolved inacetonitrile (40 mL) was added and refluxed for further 6 h (note-7).The mixture was cooled to room temperature (23° C.) and thedicyclohexylurea formed was filtered through a sintered funnel (note-8)and washed with water (3×200 mL). The filtrate was evaporated on arotary evaporator (60° C., 68-22 mbar) and dried under high vacuum atroom temperature (23° C.) (note-9). The resulting sticky syrupy residuewas redissolved in 400 mL of water to remove all dicyclohexylurea thathad remained dissolved in acetonitrile, filtered through a sinteredfunnel (note-8), and washed with water (2×100 mL). The filtrate wasevaporated on a rotary evaporator (60° C., 68-22 mbar) and dried underhigh vacuum at room temperature (23° C.). The resulting residue wasredissolved in 200 mL of water to remove any further dicyclohexylureathat had remained dissolved in solution, filtered through a sinteredfunnel (note-8), and washed with water (2×100 mL). The filtrate wasevaporated on a rotary evaporator (60° C., 68-22 mbar) and dried underhigh vacuum at room temperature (23° C.) affording hexatriethylammoniummyo-inositol 1,6:2,3:4,5 trispyrophosphate (note-10).

This hexatriethylammonium myo-inositol 1,6:2,3:4,5 trispyrophosphatesalt was dissolved in 400 mL of water, passed through a column (note-11)containing prewashed Dowex 50WX8-200 (400 g) ion exchange resin andeluted with water (4×100 mL) (note-12). To the combined acidic fractionswas immediately added solid Ca(OH)₂ (5.56 g, 0.075 mol, 1.0 eq) followedby addition of a NaOH solution [(12.0 g, 0.300 mol, 4.0 eq) in 25 mL ofwater)] at room temperature (23° C.) (note-13). Then the pH of thereaction mixture was carefully adjusted to around 6.99 with a solutionof 1:4 Ca(OH)₂:NaOH (1.5 g of Ca(OH)₂ and 3.23 g of NaOH in 1500 mL ofwater, ˜1385 mL brought the pH to ˜6.99) (note-14). Finally, the solventwas evaporated on a rotary evaporator (60° C., 68-22 mbar) and driedunder high vacuum at room temperature (23° C.) to yield the monocalciumtetrasodium myo-inositol 1,6:2,3:4,5 trispyrophosphate, ITPP Ca4Na (77.2g, 97%) as a white solid.

The compound obtained has been characterized by proton andphosphorous-31 NMR spectroscopy, mass spectroscopy, elemental analysis,cation determination by atomic absorption and water content. It containsless than 2% other phosphorous compounds. Elemental analysis (ICP atomicabsorption): P 20%; Ca 4.2%; Na 10.3% (calc.: P 25.4%; Ca 5.5%; Na12.6%). Water content: about 18-23% depending on drying conditions.

Notes:

-   -   Note-1. Purity checked in-house by ¹H and ³¹P NMR (>98%), as        well as HPLC, elemental analysis and atomic absorption for        cation determination.    -   Note-2. Collect the elutes which are acidic (pH paper). When all        phytic acid is eluted, the elute becomes neutral.    -   Note-3. Addition of triethylamine generates some heat. Add        progressively.    -   Note-4. Lower temperature can be used if evaporation can be        achieved with the equipment available.    -   Note-5. Purity and characterization was checked by ¹H and ³¹P        NMR.    -   Note-6. Reflux temperature was about 80° C. Heated with a        mantle.    -   Note-7. After 12 h, more than 98% product has been formed.        Addition of more dicyclohexylcarbodiimide led to >99% product        formation.    -   Note-8. The porosity of the sintered funnel used was 4 and this        will effectively filter off the dicyclohexylurea byproduct.    -   Note-9. Thorough drying is necessary in order to be able to        remove all the remaining dicyclohexylurea byproduct.    -   Note-10. Characterized by ¹H and ³¹P NMR, purity>99%.    -   Note-11. Two typical procedures are for instance:

Procedure 1:

The column frit porosity was 1. The diameter of the column was 8 cm andthe length of the Dowex bed was 9.5 cm. The solution was eluted first in15 minutes without any pressure and then the washings under somepressure were eluted within 5 minutes.

Procedure 2:

The column frit porosity was 2. The diameter of the column was 6 cm andthe length of the Dowex bed was 16.5 cm. The solution was eluted firstin 45 minutes without any pressure and then the washings under somepressure were eluted within 5-10 minutes.

On the basis of numerous preparations, the following is recommended:

-   -   a) a column frit porosity of 1.    -   b) a Dowex column with a height/diameter ratio of 1.5-2.0, so        that the elution time be less than 30 minutes. If the elution is        too slow then flush with some pressure.    -   Note-12. CAUTION: As the myo-inositol 1,6:2,3:4,5        trispyrophosphate free acid may hydrolyse after standing for a        long time at low pH (<1.0), the pH should be adjusted quickly to        about 3-4 in order to avoid any such hydrolysis. Check then for        absence of triethylamine signals in ¹H NMR (4-1 ppm) and for        absence of phosphorous signals around 2-4 ppm. In the unlikely        case that the ¹H NMR shows the presence of triethylamine, the        whole solution has to be passed again over a fresh Dowex-H⁺ in        order to remove it. It is very important that there be no        triethylamine left, as it would remain in the final material.    -   Note-13. It is very important to add first solid Ca(OH)₂ and        make sure that it is completely dissolved. After addition of the        NaOH solution, the pH of the reaction mixture was 1.6. The total        amount of Ca(OH)₂:NaOH required to neutralize the reaction        mixture to pH ˜6.9 was ˜6.9:14.9 g, respectively. In order to        minimize the amount of 1:4 Ca(OH)₂:NaOH solution required at the        end (and reduce the final volume), add initially 6.3 g of solid        Ca(OH)₂ followed by 13.6 g of NaOH (in 25 mL of water).        Thereafter, the amount of 1:4 Ca(OH)₂:NaOH solution required to        bring the pH to 6.9-7.0 will be significantly reduced.    -   Note-14. The 1:4 Ca(OH)₂:NaOH solution should be freshly        prepared and well closed; otherwise, CO₂ from the atmosphere        will be absorbed and insoluble materials will be formed. Adjust        the pH of the solution close to 7 and do not go beyond 7.

Example 4 Biological Activity of the Monocalcium Tetrasodium Salt ofmyo-Inositol Tripyrophosphate (ITPP-Ca4Na)

ITPP-Ca4Na acts as a powerful effector of hemoglobin shifting the oxygenfixation curves to the right, with respect to the natural effectorbisphosphoglycerate. The p50 values increase with concentration as showin FIGS. 6 and 7. In FIG. 6, hemoglobin is incubated (at concentrationsup to 100 mM final) with ITPP Ca4Na, for 1 hour at 37° C. and measuredby TCS-hemox analyzer for p50 shifts. In FIG. 7, whole human blood wasincubated (at concentrations up to 120 mM final) with ITPP Ca4Na, for 1hour 37° C. and measured by TCS-hemox analyzer for p50 shifts.

Having described the invention with reference to particularcompositions, method for detection, and source of activity, andproposals of effectiveness, and the like, it will be apparent to thoseof skill in the art that it is not intended that the invention belimited by such illustrative embodiments or mechanisms, and thatmodifications can be made without departing from the scope or spirit ofthe invention, as defined by the appended claims. It is intended thatall such obvious modifications and variations be included within thescope of the present invention as defined in the appended claims. Itshould be understood that any of the above described one or moreelements from any embodiment can be combined with any one or moreelement in any other embodiment. Moreover, when a range is mentioned, itshould be understood that it is contemplated that any real number thatfalls within the range is a contemplated end point. For example, if arange of 0.9 and 1.1 g/kg is given, it is contemplated that any realnumber value that falls within that range (for example, 0.954 to 1.052g/kg) is contemplated as a subgenus range of the invention, even ifthose values are not explicitly mentioned. All references referred toherein are incorporated by reference in their entireties. Finally, theabove description is not to be construed to limit the invention but theinvention should rather be defined by the below claims.

REFRENCES

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1. A monocalcium tetrasodium salt of inositol tripyrophosphate.
 2. Themonocalcium tetrasodium salt of inositol tripyrophosphate of claim 1,wherein the inositol tripyrophosphate is myo-inositol 1,6:2,3:4,5tripyrophosphate.
 3. A pharmaceutical composition comprising amonocalcium tetrasodium salt of inositol tripyrophosphate and apharmaceutically acceptable adjuvant, diluent, carrier, or excipientthereof.
 4. The pharmaceutical composition of claim 3, wherein theinositol tripyrophosphate is myo-inositol 1,6:2,3:4,5 tripyrophosphate.5. The pharmaceutical composition of claim 4, wherein the myo-inositol1,6:2,3:4,5 tripyrophosphate is prepared at an effective dosage fortreating cancer in a subject.
 6. The pharmaceutical composition of claim5, wherein the dosage of myo-inositol 1,6:2,3:4,5 tripyrophosphate isbetween 0.5 and 1.5 g/kg.
 7. The pharmaceutical composition of claim 5,wherein the dosage of myo-inositol 1,6:2,3:4,5 tripyrophosphate isbetween 0.9 and 1.1 g/kg.
 8. The pharmaceutical composition of claim 5,wherein the cancer is selected from the group consisting ofrhabdomyosarcomas, retinoblastoma, Ewing's sarcoma, neuroblastoma, andosteosarcoma.
 9. The pharmaceutical composition of claim 5, wherein thecancer is selected from one or more of the group consisting of breastcancer, prostate cancer, renal cell cancer, brain cancer, ovariancancer, colon cancer, bladder cancer, pancreatic cancer, stomach cancer,esophageal cancer, cutaneous melanoma, liver cancer, lung cancer,testicular cancer, kidney cancer, bladder cancer, cervical cancer,lymphoma, parathyroid cancer, penile cancer, rectal cancer, smallintestine cancer, thyroid cancer, uterine cancer, Hodgkin's lymphoma,lip and oral cancer, skin cancer, leukemia, and multiple myeloma. 10-11.(canceled)
 12. A method of treating cancer comprising administering toan individual a pharmaceutically effective amount of the composition ofclaim
 3. 13. The method of claim 12, wherein the composition isadministered to an individual at a dosage of about 0.5 to about 1.5g/kg.
 14. (canceled)
 15. A method of shifting the hemoglobin P₅₀ leveltowards higher values of oxygen partial pressure comprisingadministering to an individual an effective amount of the composition ofclaim
 3. 16. The method of claim 15, wherein the inositoltripyrophosphate is myo-inositol 1,6:2,3:4,5 tripyrophosphate.
 17. Themethod of claim 15, wherein the composition is administered at a dosageof about 0.5 to about 1.5 g/kg.
 18. (canceled)
 19. A method of treatingAlzheimer's disease comprising administering to an individual aneffective amount of the composition of claim
 3. 20. The method of claim19, wherein the inositol tripyrophosphate is myo-inositol 1,6:2,3:4,5tripyrophosphate. 21-22. (canceled)
 23. A method of enhancing thepartial pressure of oxygen in hypoxic tumors comprising administering toan individual an effective amount of the composition of claim
 3. 24. Themethod of claim 23, wherein the inositol tripyrophosphate ismyo-inositol 1,6:2,3:4,5 tripyrophosphate. 25-26. (canceled)
 27. Amethod of inhibiting angiogenesis in hypoxic tumors comprisingadministering to an individual an effective amount of the composition ofclaim
 3. 28. The method of claim 27, wherein the inositoltripyrophosphate is myo-inositol 1,6:2,3:4,5 tripyrophosphate. 29-30.(canceled)